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Flavivirus reporter virus and methods of making and using the same

a reporter virus and reporter virus technology, applied in the field of fluvirus reporter virus and methods of making and using the same, can solve the problems of limited quantitative power of plaque assays, significant challenge in vaccination against denv, and increased clinical risk of infection

Active Publication Date: 2014-04-08
INTEGRAL MOLECULAR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a method for introducing a nucleic acid molecule that encodes a flavivirus capsid polypeptide and a prME polypeptide into cells. The nucleic acid molecule can be from the same or different serotypes. The cells can then produce the capsid and prME polypeptides as a single polypeptide. The nucleic acid molecule can also be introduced into flavivirus particles, which can be used as a reporter. The invention also provides methods for identifying inhibitors of flavivirus infection and replication. Overall, this invention provides new tools for studying and manipulating flavivirus infections in cells and identifying potential inhibitors of viral infection and replication.

Problems solved by technology

The development of a vaccine for DENV has been a significant challenge and the focus of considerable effort (Monath, et al.
While antibodies play a significant role in DENV immunity, the presence of DENV antibodies has also been linked to a more severe clinical outcome due to the ability of antibodies to facilitate DENV infection under some circumstances.
The quantitative power of plaque assays is limited by the number of wells examined and the number of plaques counted by the investigator.
Thus, the PRNT approach does not allow for the neutralizing capacity of antibodies to be detected using strains that plaque poorly, or on all permissive cell types, excluding many that may be relevant in vivo.

Method used

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  • Flavivirus reporter virus and methods of making and using the same
  • Flavivirus reporter virus and methods of making and using the same
  • Flavivirus reporter virus and methods of making and using the same

Examples

Experimental program
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Effect test

example 1

[0104]Construction of a codon-optimized chimeric DENV CprME sequence. The structural genes for Dengue viruses encode for Capsid (C) Premembrane (prM) and Envelope (E) proteins (FIG. 1A). CprME sequence for Dengue serotype 2 was PCR amplified using clone 516803 with the following primers 5′-caccATGAATAACCAACGGAAAAAGGCGA-3′ (SEQ ID NO: 10) and 5′-TTTCACTATTAGGCCTGCACCATGACTCCCAAATAC-3′ (SEQ ID NO: 11). This PCR product was cloned into pENTR / D topo and subsequently moved into the expression vector pTRex-DEST-30, containing a CMV promoter and tetracycline operator sites. CprME sequence of Dengue serotype 4 was obtained by RT-PCR from RNA isolated from TVP360 virions using the following primers 5′-GGTTGATGGGCGGCCGCCACCATGAACCAACGAAAAAAGGTGGTTAGAC-3′ (SEQ ID NO: 12) and 5′-CCCTCTAGAGCGGCCTTATGCTTGAACTGTGAAGCCCAGAAACA-3′ (SEQ ID NO: 13) and was Infusion cloned as a NotI fragment into the TRex-pBR322 plasmid described below.

[0105]Plasmids containing wt DENV3 sequence are reported to be high...

example 2

[0110]Expression of DENV3 structural proteins from a codon-optimized DENV CprME sequence. DENV plasmids were used to test for expression of Dengue structural proteins in HEK-293T cells. Cells were plated in 384-well format at a density of 24,000 cells / well and transfected with plasmids encoding the CprME proteins from DENV1 wt, DENV2 wt, DENV3opt, and DENV4 wt using Lipofectamine 2000. 24 hours post transfection, cells were fixed in methanol and processed for immunofluorescence using a monoclonal antibody, 4G2 (100 μg / ml), which recognizes structural proteins of all 4 Dengue serotypes. Secondary antibody detection was with an anti-mouse Cy2 antibody. 293T cells transfected with wildtype DENV1, DENV2, and DENV4 constructs all exhibited faint staining with the 4G2 antibody. In contrast, cells transfected with the codon optimized DENV3 plasmid exhibited bright 4G2 staining, indicating that codon optimization of DENV3 results in enhanced expression of CprME in HEK-293T cells (FIG. 2).

example 3

[0111]Codon optimization of DENV2 CprME results in enhanced expression of Dengue structural proteins. Plasmids encoding wt DENV2 and codon optimized DENV2 are transfected into HEK-293T cells, or other eukaryotic cell types that can be transfected and that can express DENV proteins, to compare the expression levels of CprME. Cells are plated in 384-well format at a density of 24,000 cells / well and transfected with DENV2 wt or DENV2opt using Lipofectamine 2000. 24 hours post transfection, cells are fixed in methanol and processed for immunofluorescence using the monoclonal antibody 4G2 (100 μg / ml) with an anti-mouse Cy2 secondary antibody. HEK-293T cells transfected with DENV2 wt exhibit faint staining with the 4G2 antibody. In contrast, cells transfected with the codon optimized DENV2 plasmid exhibit bright 4G2 staining and express higher levels of CprME proteins.

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Abstract

The present invention relates to the production and uses of flavivirus replicons and flavivirus particles and reporter virus particles. The present invention relates to the production and uses of chimeric and codon-optimized flavivirus virus replicons and flavivirus virus particles and reporter virus particles.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application No. 61 / 259,279, filed Nov. 9, 2009, which is herein incorporated by reference in their entirety.GOVERNMENT SUPPORT[0002]This invention was made with U.S. Government support (NIH Grant No. AI062100) and the U.S. Government may therefore have certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Flaviviruses have a global impact due to their widespread distribution and ability to cause encephalitis in humans and economically important domesticated animals. Of the approximately seventy viruses in the genus, roughly half have been associated with human disease. Several members of this group, such as dengue virus (DENV) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because the incidence with which they encounter humans and cause disease is increasing each year at an alarming rate. Globally, DENV has become the most significant source of ar...

Claims

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Application Information

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IPC IPC(8): C07H21/00A61K39/12
CPCC12N2770/24151C12N2800/22C07H21/00C12N2830/003C12N15/86A61K39/12C12N15/00C12N2770/24143C12N7/00
Inventor PUFFER, BRIDGETMATTIA, KIMBERLY-ANNE
Owner INTEGRAL MOLECULAR
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